Biochemical and structural characterisation of a protozoan beta-carbonic anhydrase from Trichomonas vaginalis

Abstract

We report the biochemical and structural characterisation of a beta-carbonic anhydrase (β-CA) from Trichomonas vaginalis, a unicellular parasite responsible for one of the world's leading sexually transmitted infections, trichomoniasis. CAs are ubiquitous metalloenzymes belonging to eight evolutionarily divergent groups (α, β, γ, δ, ζ, η, θ, and ι); humans express only α-CAs, whereas many clinically significant pathogens express only β- and/or γ-CAs. For this reason, the latter two groups of CAs are promising biomedical targets for novel antiinfective agents. The β-CA from T. vaginalis (TvaCA1) was recombinantly produced and biochemically characterised. The crystal structure was determined, revealing the canonical dimeric fold of β-CAs and the main features of the enzyme active site. The comparison with the active site of human CA enzymes revealed significant differences that can be exploited for the design of inhibitors selective for the protozoan enzyme with respect to the human ones.

Keywords: Beta carbonic anhydrase; Trichomonas vaginalis; crystal structure; kinetics; protozoan.

Figure 1.

Illustration of the pBVboostFG expression vector. The designed parts of the insert: 1. attL1, 2. Shine-Dalgarno, 3. Kozak, 4. Met-Ser-Tyr-Tyr, 5. 6 × His, 6. Asp-Tyr-Asp-Ile-Pro-Thr-Thr, 7. Lys-Val, 8. CA gene of interest, 9. 2 × stop codon, 10. attL2.

Figure 2.

SDS-PAGE of purified TvaCA1 with a 6xHis-tag (lane 1) and after removal of the tag (lane 2). All the polypeptide bands shown on the gel were identified as TvaCA1 protein by MS/MS. The standard molecular weight (M_w) marker is shown on the far left.

Figure 3.

Light scattering data for the assessment of the oligomeric state and size of TvaCA1. The left Y-axis shows the UV absorption intensity at 280 nm and right-angle light scattering intensity (RALS). The right Y-axis shows the M_w calculated using static LS intensity.

Figure 4.

(A) Ribbon representation of the TvaCA1 monomer. (B) Enlarged view of the active site, showing Zn²⁺ coordination. (C) σA-weighted |2Fo-Fc| electron density map (contoured at 1.0 σ) relative to zinc ion coordination site.

Figure 5.

Dimeric structure of TvaCA1, with one monomer coloured in magenta and the other in green. The catalytic zinc ions are depicted as yellow spheres.

Figure 6.

Surface representation of (A) hCA II, chosen as a representative hCA isoform, and (B) TvaCA1. Residues delimiting the rim of the active site cavity are coloured in red. The metal ions are shown as yellow spheres. It is evident that in hCA II, the active site rim is larger (approximately 15 Å × 14 Å) and more accessible than that in TvaCA1 (8 Å × 6.5 Å).